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D A Ritchie

Publications and source records attributed to D A Ritchie.

At least 91 records · Page 5Linked to original sources
PubMed1984

Phage head assembly in bacteriophage T1.

Phage-related structures found in wild-type and mutant T1 infections were examined by sedimentation analysis, electron microscopy, and polyacrylamide gel electrophoresis. Phage-particle polypeptides P7 (molecular weight 33,000) and P11 (molecular weight 16,000) were identified as major head proteins and P10 (molecular weight 26,000) was shown to be the major tail protein. A DNA-free head-like structure containing P7 but not P11 was present in wild-type and a number of mutant infections. In view of its possible role as a precursor to mature heads, this structure was termed the prohead. Mutants in two genes, am10 (gene 13) and am45 (gene 14), synthesised only tails in nonpermissive infections. Mutants in eight of the head genes, am23 (gene 4), am283 (gene 13.3), am216 (gene 13.7), ts257 (gene 14.5), am11 (gene 15), am4 (gene 16), am7 (gene 17), and am30 (gene 18), synthesised proheads and tails but not other structures. am37 (gene 12) synthesised proheads, tails, and empty heads. Studies with a tail-defective mutant suggested that empty heads were produced from the breakdown of DNA-filled heads. P11, the T1 gene 13.3 product, is analogous to the phage lambda gene D protein: both are major capsid proteins which appear to stabilise the later stages of head filling. These data are reviewed according to the general principles established for phage-head assembly and a tentative pathway for T1-head assembly is proposed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1984

The structure of replicating bacteriophage T1 DNA: comparison between wild type and DNA-arrest mutant infections.

Analysis of the structure of replicating phage T1 DNA has identified three major forms of T1+ intracellular DNA; (i) concatemeric molecules with single-stranded interruptions, (ii) monomer-length DNA with single-strand interruptions, and (iii) monomer-length molecules with completely intact single strands. The interruptions in concatemeric DNA are spaced at intervals, with a mean distance equivalent to one monomer length but with a broad distribution and many are in the form of gaps. Type (iii) molecules are probably derived from mature progeny phage particles disrupted during cell lysis. Under nonpermissive conditions, cells infected with amber mutants in T1 genes 3.5 and 4 show the premature arrest of phage DNA synthesis, failure to make concatemeric DNA, and reduced genetic recombination. Intracellular DNA from gene 3.5- and gene 4- infections consist of a uniform population of molecules approximately 10-12% shorter than mature monomers. They are stable throughout infection, contain single-stranded interruptions but not gaps, and are missing terminal sequences. These results are interpreted in terms of concatemer formation by end-to-end recombination between newly synthesised molecules whose terminal sequences are degraded in recombination-defective infections.

Base Sequence↗
PubMed1984

Formation of phage T1 concatemers by the RecE recombination pathway of Escherichia coli.

Infections of nonpermissive ( sup0 ) Escherichia coli by T1 phage with amber mutations in either gene 3.5 or gene 4 exhibit a variety of defective phenotypes, including premature arrest of T1 DNA synthesis, failure to make concatemeric DNA, formation of an abnormal DNA replication intermediate, failure to package phage DNA, and reduced genetic recombination. The lethal effect of gene 3.5 or 4 mutations is suppressed when the sup0 bacteria express the RecE recombination pathway. This RecE suppression occurs by partial restoration of the capacity to make concatemeric molecules and partial reversal of the DNA arrest defect which, in turn, leads to the formation of viable progeny. Infection by T1+ or by mutants defective in any of the four DNA synthesis genes (genes 1, 2, 3.5, and 4) inhibited the ATP-dependent exonuclease present in uninfected cells (presumably the RecBC enzyme, exonuclease V). Extracts from T1+ infections also showed increased levels of an ATP-independent exonuclease activity which was absent from gene 4 mutant extracts. It is concluded that gene 4, together with gene 3.5, specifies an activity related to that of the RecE exonuclease VIII and essential for T1 concatemer formation and recombination.

DNA Replication↗
PubMed1983

Uncoupling of initiation site cleavage from subsequent headful cleavages in bacteriophage T1 DNA packaging.

The packaging of intracellular DNA into heads is a key feature in the morphogenesis of bacteriophage particles. In many phages a performed empty head precursor, the prohead, is filled with DNA from a concatemeric substrate consisting of tandemly repeated genome lengths. The addition of outer shell proteins completes head formation. The DNA molecules released from particles of the coliphage T1 exist as three major permutations of nucleotide sequence. Such limited permutation can be explained by the modification of Streisinger's 'headful' mechanism proposed for phage P22. DNA packaging is initiated at a specific site (the pac site) on the concatemeric precursor. While this site is cleaved, subsequent cleavages (headful cleavages) are dependent only on head-filling and are not defined in terms of nucleotide sequence. Headfuls of DNA, consisting of slightly more than a genome length, are packaged in three successive cycles of head-filling to produce the permuted and terminally redundant molecules characteristic of T1 DNA. To elucidate the regulation of this process, we have studied the DNA metabolism of T1 head mutants. We describe here the properties of a mutant in gene 13.3 which is defective for headful cleavage but remains proficient in pac site cleavage. The observation in this mutant that concatemers are degraded to unit-length molecules by repeated pac site cleavage suggests a model of headful packaging in which pac site initiation and processive head-filling compete for the DNA substrate.

Bacterial Proteins↗
PubMed1983

Correlation of genetic loci and polypeptides specified by bacteriophage T1.

The effects of amber mutations in the 24 known essential genes of phage T1 on phage-directed protein synthesis were examined by SDS-polyacrylamide gel electrophoresis of radiolabelled polypeptides from infected non-permissive cells. Mutations in nine genes (genes 1, 2, 3, 3.5, 5, 7-8, 10, 13.3 and 15) each resulted in the failure to synthesize a single polypeptide. Since the synthesis of each of these polypeptides was at least partially restored in permissive infections and wholly restored in am+ revertant infections we conclude that the affected polypeptide is the primary gene product. Mutations in genes 13.7, 16 and 17, which are required for head formation, are all defective in the synthesis of a 68000 mol. wt. non-structural polypeptide. Mutants in the head genes 13 and 14 fail to cleave P7p to P7, the major structural component of proheads. Gene 14 mutants also fail to make a 45000 mol. wt. non-structural polypeptide which appears to be involved with the gene 13 product in the P7p cleavage reaction. The protein products of the DNA genes 1, 2 and 3.5 are, as expected, synthesized predominantly early in infection whereas those of the remainder, which determine head and tail formation, are made in greater amounts late in infection. Gene 3, a tail gene which has been mapped within the 'early' DNA gene cluster, codes for a 75000 mol. wt. polypeptide synthesized predominantly late in infection. This observation suggests that the early genes 1, 2, 3.5 and 4 do not form an operon under the control of a single early promoter.

Electrophoresis, Polyacrylamide Gel↗
PubMed1980

A physical map of the permuted genome of bacteriophage T1.

A restriction map has been constructed for the DNA of coliphage T1 which locates the cleavage sites of the restriction endonucleases, BglI (6 cuts), BglII (16 cuts), EcoRI (2 cuts), HindIII (2 cuts) and PstI (2 cuts). Digestions with BglI and BglII reveal fragments which are present in sub-molar quantities. Two methods, one using the selective removal of molecular ends with exonuclease III and the other involving the comparison of digestion patterns of concatemeric and virion DNA, have shown that the submolar fragments are at or close to the ends of the molecules. Digestions with BglI show that one terminal fragment has a very precise molecular weight whereas all the others are of heterogenous molecular weight. These results are consistent with the model for DNA packaging in which maturation is initiated at a precise site on a concatemeric precursor and proceeds by the encapsidation of up to four successive 'headfuls' of 1.065 genome equivalents (MacHattie and Gill 1977).

Chromosome Mapping↗
PubMed1978

Formation of concatemeric DNA as an intermediate in the replication of bacteriophage T1 DNA molecules.

The structure of intracellular DNA extracted from phage T1 infected cells was analysed by sedimentation through sucrose gradients. DNA labelled with 3H-dThd during a short pulse given at any time during T1 DNA synthesis sedimented in neutral gradients as a broad heterogeneous band with a large fraction of the label sedimenting more rapidly than mature T1 DNA molecules. Rapidly-sedimenting label was also observed when pulse-labelled DNA was denatured and analysed on alkaline sucrose gradients. Electron microscopy of intracellular T1 DNA revealed linear molecules of variable length the longest of which were three to four times the mature genome length. The distribution of lengths derived from electron microscopy are consistent with the molecular length distributions calculated from the sedimentation coefficients. We conclude that the rapidly-sedimenting DNA is in the form of concatemers consisting of linear tandem repeats of the T1 genome. The concatemeric form of replicating T1 DNA is a precursor of progeny T1 genomes since in pulse-chase experiments it was converted efficiently into mature, infectious T1 phage particles. The identification of this concatemeric form of T1 DNA provides supporting evidence for the model proposed by Gill & MacHattie (1976) to account for the formation of the very limited number of cyclic permutations of gene sequence found for mature T1 DNA molecules.

Centrifugation, Density Gradient↗
PubMed1976

Polypeptides specified by bacteriophage T1.

The proteins synthesized during the replication of phage TI in u.v.-irradiated Escherichia coli strain B have been examined by polyacrylamide gel electrophoresis of polypeptides pulse-labelled with 14C-amino acids. Up to 50 discrete bands were identified of which about 30 were sufficiently distinct to be classified in terms of time of synthesis. Three polypeptides were synthesized only during the first 6 to 8 min post infection (Class I, Early); 16 or 17 were synthesized predominantly during the later stages of replication starting from 6 to 8 min after infection (Class III, Late); three classes of proteins were made continuously, two at constant rate (Class II, Continuous), five at decreasing rate (Class IV, Early-continuous) and five at increasing rate (Class V, Late-continuous). Of the 14 polypeptides identified as structural components of the virion, three (P7, PI0 and PI) account for about 85% of the particle weight with P7 comprising 50% of the particle. P7 and PI0 appear to result from the cleavage of larger polypeptides. Preliminary studies with amber mutants suggest that normal levels of TI DNA synthesis are not required for the manufacture of late proteins and that a phage-controlled function may control the switch-off of proteins made early and the switch-on of proteins made late.

Coliphages↗
PubMed1975

The induction of mutants of Acinetobacter calcoaceticus NCIB8250 and their selection by vancomycin.

The mutagenic and lethal effects of N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulphonate (EMS), ultraviolet light irradiation and near-ultraviolet light irradiation with 8-methoxypsoralen on the bacterium Acinetobacter calcoaceticus NCIB8250 were examined. The production of auxotrophic mutants was used as a measure of mutagenic efficiency. Under appropriate conditions all four agents were mutagenic. EMS and NTG although more effective than irradiation, did not cause such a high frequency of mutation as has been observed with other bacteria. A combination of vancomycin and penicillin V gave enrichment of non-metabolizing bacteria and optimum conditions were found for the use of these compounds in a selection technique.

Acinetobacter↗